Thrombin is a serine protease belonging to the trypsin family. It is a key enzyme involved in the blood coagulation system.
The coagulation system can be activated in response to surface contact or vascular injury, through either an intrinsic pathway or an extrinsic pathway, respectively. In the intrinsic pathway, all of the protein components involved in coagulation are present in blood. In the extrinsic pathway, by contrast, cell membrane proteins (i.e., tissue factors) are involved in, and play, a critical role.
Typically, the coagulation system is activated in response to vascular injury. The last two steps of the coagulation cascade leading to clot formation are common to both pathways. The first of these two steps involves the conversion of prothrombin to thrombin. This conversion is catalyzed by the enzyme Factor Xa. Thrombin then converts fibrinogen to insoluble fibrin, which is a major component of blood clots.
In addition to thrombin's role in blood coagulation, the formation of thrombin at a site of vascular injury also activates numerous cells involved in inflammatory reactions and tissue reparative processes. Such cells include monocytes, T lymphocytes, fibroblasts, endothelial cells and mast cells. For example, it is reported that thrombin stimulates adhesion and aggregation of platelets, activation of endothelial cells, release of growth factors from cells, and adhesion and recruitment of leukocytes, such as monocytes and T lymphocytes.
Abnormalities in the coagulation cascade have been implicated in numerous vascular diseases and conditions. Such diseases and conditions include cardiovascular diseases (e.g., thrombosis such as deep vein thrombosis, myocardial thrombosis, etc., and peripheral arterial occlusion, coronary artery disease, myocardial ischemia, pulmonary embolism, etc.), cerebrovascular diseases (e.g., stroke such as ischemic stroke and thrombotic stroke). These diseases and conditions are generally characterized by either partial or total occlusion of a blood vessel by a blood clot.
In order to prevent or treat conditions and disorders associated with abnormal coagulation, therapeutic methods to inhibit clot formation or to dissolve clots have been developed. Existing anticoagulants, however, produce side effects. For example, heparin is a widely used anticoagulant drug. Heparin administration, however, can cause bleeding and thrombocytopenia (i.e., decrease in platelets). In addition, heparin has to be injected or infused and the half-life in the circulation is short.
Another class of anticoagulants is that of the coumarins, of which coumadin (referred to generically as warfarin) is commonly used. However, a disadvantage of warfarin is that it takes several days to achieve its maximum effect. As with heparin, bleeding can also be a complication. In addition, warfarin is teratogenic, and can cross the placenta, causing fetal abnormalities when administered to pregnant women.
Thrombolytic agents, which dissolve existing clots, are also used therapeutically. Their activity is based on enhancing the generation of plasmin from its plasminogen precursor. The enzyme plasmin generally dissolves blood clots by specifically cleaving fibrin.
Such thrombolytic agents include recombinant tissue plasminogen activator (tPA) and streptokinase. Disadvantages of these thrombolytics include a systemic fibrinolytic activity that can result in bleeding throughout the body. Further, some thrombolytics (e.g., streptokinase) are antigenic and can cause allergic reactions.
The compound tetracycline is a member of a class of antibiotic compounds that is referred to as the tetracyclines, tetracycline compounds, tetracycline derivatives and the like. The compound tetracycline exhibits the following general structure:
The numbering system of the tetracycline ring nucleus is as follows:

Tetracycline, as well as the terramycin and aureomycin derivatives, exist in nature, and are well known antibiotics. Natural tetracyclines may be modified without losing their antibiotic properties, although certain elements must be retained. The modifications that may and may not be made to the basic tetracycline structure have been reviewed by Mitscher in The Chemistry of Tetracyclines, Chapter 6, Marcel Dekker, Publishers New York (1978). According to Mitscher, the substituents at positions 5-9 of the tetracycline ring system may be modified without the complete loss of antibiotic properties.
In addition to their antibacterial properties, tetracyclines have been described as having a number of other uses. For example, tetracyclines are also known to inhibit the activity of collagen destructive enzymes, produced by mammalian (including human) cells and tissues, by non-antibiotic mechanisms. Such enzymes include the matrix metalloproteinases (MMPs), including collagenases (MMP-1, MMP-8 and MMP-13), gelatinases (MMP-2 and MMP-9), and others (e.g., MMP-12, MMP-14). See Golub et al., J. Periodont. Res. 20:12-23 (1985); Golub et al. Crit. Revs. Oral Biol. Med. 2:297-322 (1991); U.S. Pat. Nos. 4,666,897; 4,704,383; 4,935,411; 4,935,412. Also, tetracyclines have been known to inhibit wasting and protein degradation in mammalian skeletal muscle, U.S. Pat. No. 5,045,538; to inhibit inducible NO synthase, U.S. Pat. Nos. 6,043,231 and 5,523,297; to inhibit phospholipase A2, U.S. Pat. Nos. 5,789,395 and 5,919,775; to inhibit neutrophil elastase, U.S. Pat. No. 5,773,430; and to enhance IL-10 production in mammalian cells, U.S. Pat. No. 6,015,804. These properties cause the tetracyclines to be useful in treating a number of diseases.
It is one object of the present invention to provide a method for inhibiting undesirable thrombin generation. Inhibition of undesirable thrombin generation is useful in diseases and conditions associated with abnormal blood clotting.